KINETIC EVIDENCE FOR THE EXISTENCE OF A RATE-LIMITING STEP IN THE REACTION OF FERRIC HEMOPROTEINS WITH ANIONIC LIGANDS

The kinetics of azide and fluoride binding to various monomeric and te trameric ferric hemoproteins (sperm whale Mb, isolated alpha and beta chains of human Hb reacted with p-chloromercuribenzoate, dromedary, ox and human Hb) has been investigated (at pH 6.5 and 20 degrees C) over a large range (20 mu M to 2 M) of ligand concentration. It has been o bserved that the pseudo-first-order rate constant for azide binding to the hemoproteins investigated does not increase linearly with ligand concentration, but tends to level off toward an asymptotic concentrati on-independent value typical for each hemoprotein. This behaviour, whi ch has been detected only by an investigation covering an unusually la rge range of ligand concentrations, appears to be independent of the i onic strength, and it underlies the existence of a rate-limiting step in the dynamic pathway of azide binding to ferric hemoproteins, which is detectable whenever the observed pseudo-first-order rate constant b ecomes faster than a given value characteristic of the specific hemopr otein. Such a behaviour is not observed in the case of fluoride bindin g probably because the pseudo-first-order rate constant for this ligan d is much slower and never attains a value faster than that of the rat e-limiting step. In general terms, this feature should involve a confo rmational equilibrium between at least two forms (possibly related to the interaction of H2O with distal histidine and its exchange with the bulk solvent) which modulates the access of the anionic ligand into t he heme pocket and its reaction with the ferric heme iron.